Boron neutron capture therapy (BNCT) is a binary cancer treatment modality where two different agents (10B and thermal neutrons) have to be present to produce an effect. A dedicated trial design is necessary for early clinical trials. The concentration of 10B in tissues is an accepted surrogate to predict BNCT effects on tissues. Tissue, blood, and urines were sampled after infusion of two different boron carriers, namely BSH and BPA in the frame of the European Organisation for Research and Treatment of Cancer (EORTC) trial 11001. In this study, urine samples were used to identify protein profiles prior and after drug infusion during surgery. Here, an approach that is based on the mass spectrometry (MS)-based proteomic analysis of urine samples from head and neck squamous cell carcinoma (HNSCC) and thyroid cancer patients is presented. This method allowed the identification of several inflammation- and cancer-related proteins, which could serve as tumor biomarkers. In addition, changes in the urinary proteome during and after therapeutic interventions were detected. In particular, a reduction of three proteins that were involved in inflammation has been observed: Galectin-3 Binding Protein, CD44, and osteopontin. The present work represents a proof of principle to follow proteasome changes during complex treatments based on urine samples.

As suggested by the title of this Special Issue, liquid chromatography-mass spectrometry plays a pivotal role in the field of proteomics. Indeed, the research and review articles published in the Issue clearly evidence how the data produced by this sophisticated methodology may promote impressive advancements in this area. From among the topics discussed in the Issue, a few point to the development of new procedures for the optimization of the experimental conditions that should be applied for the identification of proteins present in complex mixtures. Other applications described in these articles show the huge potential of these strategies in the protein profiling of organs and range from to the study of post-translational tissue modifications to the investigation of the molecular mechanisms behind human disorders and the identification of potential biomarkers of these diseases.

Background Infectious Leptospira colonize the kidneys of reservoir (e.g. rats) and accidental hosts such as humans. The renal response to persistent leptospiral colonization, as measured by urinary protein biosignatures, has not been systematically studied. Urinary exosomes--bioactive membrane-bound nanovesicles--contain cell-state specific cargo that additively reflect formation all along the nephron. We hypothesized that Leptospira-infection will alter the content of urine exosomes, and further, that these Leptospira-induced alterations will hold clues to unravel novel pathways related to bacterial-host interactions. Methodology/Principal findings Exosome protein content from 24 hour urine samples of Leptospira-infected rats was compared with that of uninfected rats using SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC-MS/MS). Statistical models were used to identify significantly dysregulated proteins in Leptospira-infected and uninfected rat urine exosomes. In all, 842 proteins were identified by LC-MS/MS proteomics of total rat urine and 204 proteins associated specifically with exosomes. Multivariate analysis showed that 25 proteins significantly discriminated between uninfected control and infected rats. Alanyl (membrane) aminopeptidase, also known as CD13 topped this list with the highest score, a finding we validated by Western immunoblotting. Whole urine analysis showed Tamm-Horsfall protein level reduction in the infected rat urine. Total urine and exosome proteins were significantly different in male vs. female infected rats. Conclusions We identified exosome-associated renal tubule-specific responses to Leptospira infection in a rat chronic colonization model. Quantitative differences in infected male and female rat urine exosome proteins vs. uninfected controls suggest that urine exosome analysis identifies important differences in kidney function that may be of clinical and pathological significance.

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors.

Ginkgo biloba is an ancient Chinese tree, appeared more than 250 million years ago, and the only surviving member of Ginkgoacea family [Schmid, 1997]. Ginkgo biloba was used as herbal remedy for many centuries in China, and now its extracts are one of the most widely used herbal products in the world, especially in the United States and in Europe [Blumenthal, 2000; Mahadevan & Park, 2008]. Ginkgo biloba extract is considered an alternative medicine for the treatment and/or the prevention of different pathologies and in some cases it could be suggested to be used as complementary of the mainstream medicine [Ernst, 2000]. In fact, over the past decades, there was a steady growth trend in the use of these alternative treatments. In particular, concentrated and partially purified products, containing Ginkgo biloba active constituents, have been marketed widely in the world for the treatment of cognitive deficits and other age-associated impairments [Kanowski et al., 1996; Le Bars et al., 1997]. Furthermore, it has been used as therapeutic compound for many other chronic and acute forms of diseases such as cardiovascular and bronchial pathologies [Diamond et al., 2000]. In view of the large market as well as the keen interest in the use and rediscovery of these herbal products throughout the world, the quality control of Ginkgo biloba extracts becomes necessary, in order to guarantee their clinical efficacy and safety. Therefore, it is important to monitor simultaneously the bioactive constituents present in Ginkgo biloba extracts, optimizing the analysis time and reducing costs. In fact, in the recent years, numerous groups reported in literature different analytical methods, using various chromatographic conditions and spectophotometric technologies, to create quick, accurate and applicable analytical approaches for the identification and the chemical structure characterization of Ginkgo biloba constituents. Ginkgo biloba extracts contain a large number of representative constituents such as terpenoids, polyphenols, allyl phenol, organic acids, carbohydrates, fatty acids and lipids, inorganic salts and amino acids. However, the pharmacological activity of Ginkgo biloba

In the mouse, the antral compartment of the ovary contains two different types of oocytes (SN and the NSN) distinguishable by a different transcriptional activity and developmental competence: only the SN types maturated and fertilized in vitro are able to develop until the blastocyst stage. Since the functional meaning of SN and NSN oocytes is still highly debated, we used a multi-approach analysis employing molecular, biophysics and biochemical methods to understand the molecular events responsible for the two different chromatin organizations and thus for the two different prospective zygotic developments.Gene expression analyses suggested that the NSN-derived zygotic epigenome shows reduced levels of expression of some important genes involved in cell differentiation and transcription. For instance, the transcription factor Oct4 is detected with low levels of expression in the NSN compared to the SN type: this might explain the greater developmental capacity of the SN oocytes and its correlation with the totipotent characteristic of the future zygote. Using microarrays technology we characterized global changes in gene expression in SN versus NSN oocytes showing an over-expression of many early response genes often associated with stress and apoptosis together with many genes involved in the translation and polyadenylation process.To get new insights on the molecular composition and organization of SN and NSN oocytes at the GV stage and during the transition MI-MII stage, we recently used Fourier transformed infrared microspectroscopy, showing important differences in the protein absorption regions concerning the extent of transcription, polyadenylation and methylation. The NSN oocytes maintain, during the maturation process, a higher transcriptional activity than the SN type. At MII, SN-derived oocytes showed a significant polyA content that could reflect the storage of maternal mRNAs with polyA tails, crucial for the subsequent early embryonic development, while the NSN-derived oocytes lacking a properly polyadenylated maternal mRNAs, are kept in an unsuccessful transcriptional state. However, the most evident separation between the two types of oocytes is at the MI stage, considered a crucial checkpoint where some of the molecules and/or mechanisms rearrange to then participate in the decision of oocyte fate and destiny towards, or not, meiotic resumption.NSN oocytes reflect also a partially methylated DNA (indicated by a reduction in the CpG island methylation) and enter into a transcriptional standby state at the MI stage. The maturation of the NSN oocytes is characterized by a higher transcriptional activity at MII than at the antral stage and by a lower level of polyadenylation of mRNAs. In addition, we are currently identifying the protein profiles of the two different oocytes by means of MudPIT (Multidimensional Protein Identification Technology), a gel-free approach based on two dimensional micro-chromatography coupled to tandem mass spectrometry (2DC-MS/MS). By doing this, hundreds of proteins will be identified for each sample and differentially expressed proteins determined by comparing SN versus NSN oocytes.Taken together these results will allow us to better comprehend the mechanisms involved in the generation of developmentally fully competent oocytes in vitro and in the reliable genetic control of mammalian reproduction. (poster)

Ginkgo biloba is an ancient Chinese tree, appeared more than 250 million years ago, and the only surviving member of Ginkgoacea family [Schmid, 1997]. Ginkgo biloba was used as herbal remedy for many centuries in China, and now its extracts are one of the most widely used herbal products in the world, especially in the United States and in Europe [Blumenthal, 2000; Mahadevan & Park, 2008]. Ginkgo biloba extract is considered an alternative medicine for the treatment and/or the prevention of different pathologies and in some cases it could be suggested to be used as complementary of the mainstream medicine [Ernst, 2000]. In fact, over the past decades, there was a steady growth trend in the use of these alternative treatments. In particular, concentrated and partially purified products, containing Ginkgo biloba active constituents, have been marketed widely in the world for the treatment of cognitive deficits and other age-associated impairments [Kanowski et al., 1996; Le Bars et al., 1997]. Furthermore, it has been used as therapeutic compound for many other chronic and acute forms of diseases such as cardiovascular and bronchial pathologies [Diamond et al., 2000]. In view of the large market as well as the keen interest in the use and rediscovery of these herbal products throughout the world, the quality control of Ginkgo biloba extracts becomes necessary, in order to guarantee their clinical efficacy and safety. Therefore, it is important to monitor simultaneously the bioactive constituents present in Ginkgo biloba extracts, optimizing the analysis time and reducing costs. In fact, in the recent years, numerous groups reported in literature different analytical methods, using various chromatographic conditions and spectophotometric technologies, to create quick, accurate and applicable analytical approaches for the identification and the chemical structure characterization of Ginkgo biloba constituents. Ginkgo biloba extracts contain a large number of representative constituents such as terpenoids, polyphenols, allyl phenol, organic acids, carbohydrates, fatty acids and lipids, inorganic salts and amino acids. However, the pharmacological activity of Ginkgo biloba

For a good clinical outcome of Haemophilia A substitutive therapy a detailed characterization of factor VIII (FVIII) concentrates is required, in order to disclose the eventual relations between differently composed concentrates and their biological effects. This preliminary work could be a first step towards a deep structural characterization of FVIII concentrates, using the fast and simply manageable MudPIT technology, which enables the identification and characterization of protein mixtures taking advantage of both the high separation capacity of two-dimensional chromatography and the powerful peptide characterization ability of tandem mass spectrometry. The aim of this study was to evaluate the suitability of for the characterization of FVIII molecule in complex mixtures such its commercial concentrates, both plasma-derived and recombinant, and for the determination of the protein composition of different FVIII preparations. By means of Multidimensional Protein Identification Technology (MudPIT) it was possible to assess the presence of factor VIII in its preparations and to identify most of the contaminant proteins without gel separation. In particular, 125 and 42 proteins were identified in plasma-derived and recombinant concentrates, respectively. Concerning investigation of FVIII, 24 different peptides were identified in plasma-derived corresponding to 7, 29, 27, 19 and 67 of percentage coverage for A1, A2, A3, C1 and C2 domains, respectively. About its multimeric carrier von Willebrand factor (VWF), we have sequenced 42% of domain interacting with A3 and C2 domains of FVIII. Finally, it has been observed that normalized parameters, such as total peptide hits obtained by SEQUEST may be used for evaluation of the relative abundance of FVIII in different preparations.

Within the clinical trial EORTC 11001, patients were infused with (10)B-enriched borono-phenylalanine-fructose complex (BPA-fr), or borocaptate sodium (BSH) solutions, which are used as boron carriers for boron neutron capture therapy. Urine samples were periodically collected and analyzed by (10)B NMR spectroscopy. The results revealed time-dependent metabolic changes of the administered compounds. BPA-fr dissociated to the constituents BPA and fructose, and the borate group was partly cleaved from BPA. BSH was partly aggregated to a dimer form, BSSB. These observations were previously reported for cultured cells and animal models, and are confirmed here in human cancer patients.

Boron Neutron Capture Therapy (BNCT) is a radiotherapic technique still under development that could become crucial in the fight against some types of cancer (extended ones, located near vital organs or radioresistant). This binary technique requires the administration to the patient of a boron delivery agent and the irradiation with a thermal neutron beam. The high LET particles produced in the B-10(n, alpha)Li-7 reaction are exploited to destroy the tumour cells. This work presents a new system based on neutron autoradiography with a non-depleted self-triggering microstrip silicon detector, using a neutron beam produced by a hospital Linac. The system is fast, real time and allows the detection of B-10 contents down to 25 ng. The main results on the study of B-10 uptake in biological samples will be described in terms of kinetic curves (B-10 uptake as a function of time).

Standardized extracts of Ginkgo biloba L. leaves are widely used in clinical practice for the symptomatic treatment of mild to moderate dementia syndromes, cerebral insufficiency and for the enhancement of cognitive function. The main active components present in G. biloba extracts are flavonol-glycosides and terpene-lactones. In recent investigations, the sesquiterpene trilactone bilobalide has been described to exert an interesting neuroprotective effect when administered systemically to experimental animals. Oral administration of terpene-lactones either as standardized extracts or purified products is characterized by a low bioavailability. While preparing phospholipidic complex of G. biloba extracts or bilobalide, plasma levels of terpenes and sesquiterpene increase. In the present study, phospholipidic complex of bilobalide (IDN 5604) has been administered orally to rats and bilobalide levels have been determined in plasma and brain by means of a validated method based on liquid chromatography coupled to atmospheric pressure chemical ionization ion trap mass spectrometry (LC/APCI-ITMS). Due to its sensitivity (about 3 pmol/ml) and specificity, LC/APCI-ITMS method proved to be a very powerful tool for pharmacokinetic studies of Ginkgo terpene-lactones. The results of the present study clearly confirm the improvement of oral bioavailability of bilobalide administered as phospholipidic complex and, for the first time, demonstrate the detection of significative amounts of bilobalide in brain. This last finding agrees with the neuroprotective activity observed for bilobalide.

Different fractionation steps have been developed to reduce the complexity of serum proteome and to allow the detection and the identification of single proteins. The nature of fractionation ( performed either on denatured or native proteins), the efficiency of recovery, the capacity of directly identifying proteins or protein panels, the possibility of associating to other laboratory techniques influence the choice of the methods to be used in different experimental and clinical settings. In this review, the main fractionation techniques (such as electrophoresis, SELDI and liquid chromatography) are described for proteomic studies in clinical field, and their advantages and disadvantages are discussed on the basis of our experience. In particular, liquid-liquid fractionation performed by PF2D is discussed in detail because of its partial automation, its ability to improve mass spectrometry analysis of serum proteins and its potential application in clinical studies.

Using agents for developing intrusion detection systems can provide several advantages, including configurability, adaptability, scalability, and robustness. Almost all works in agent-based intrusion detection have considered agents as elements that perform specific tasks in the intrusion detection process. In this paper, we propose a novel way of using agents to solve one of the most pressing problems in intrusion detection: the definition of an accurate model of network normality. We consider agents as associated to partial models of network normality that harmonize their conflicts via cooperative negotiation. Experimental results show that the proposed approach is promising.

Liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry (LC/APCI-ITMS) was applied to determine the concentration of terpene lactone in plasma of guinea pigs after chronic administration of Ginkgo biloba extract enriched in ginkgoterpenes in free form (IDN 5380) or complexed with soy phosphlipids (IDN 5381). Oral treatment of the animals with ginkgoterpenes resulted to inhibit the bronchoconstriction (ITP) and the concomitant increase of the levels of thromboxane B2 (TXB2) in the circulation caused by histamine (HIST) and platelet activating factor (PAF) in normal guinea pigs or by ovalbumin (OA) in actively sensitized guinea pigs. To compare the protective activities of G. biloba forms (IDN 5380 and IDN 5381), ED50 and dose ratio (DR) values for both parameters (ITP and TXB2) were evaluated. The phytosomic form (IDN 5381) significantly reduced (two- to four-fold as compared to free form, P < 0.001) the HIST, PAF or OA-induced airway changes and TXB2 release. In addition it has been observed that the absence of ginkgolide C (GC) in plasma samples (in human and animals) was due to its rapid methylation.